NF-Y substitutes H2A-H2B on active cell-cycle promoters: recruitment of CoREST-KDM1 and fine-tuning of H3 methylations.
The CCAAT box is a frequent promoter element, as illustrated by bioinformatic analysis, and it is bound by NF-Y, a trimer with H2A-H2B-like subunits. We developed a MNase I-based ChIP protocol on homogeneous cell populations to study cell-cycle promoters at the single nucleosome level. We analyzed histone methylations and the association of enzymatic activities. Two novel results emerged: (i) H3-H4 are present on core promoters under active conditions, with the expected cohort of 'positive' modifications; H2A-H2B are removed and substituted by NF-Y. Through the use of a dominant negative mutant we show that NF-Y is important for H3K36me3 deposition and for elongation, not recruitment of Pol II; (ii) H3K4 methylations are highly dynamic and H3K4me1 is a crucial positive mark. Functional siRNA inactivation and treatment with Tranylcypromine determined that KDM1 (LSD1) plays a positive role in transcription, specifically of G2/M genes. It requires CoREST, which is recruited on active promoters through direct interactions with NF-Y. These data are the first in vivo indication of a crucial interplay between core histones and 'deviant' histone-fold such as NF-Y, leading to fine-tuning of histone methylations.
Gatta
Raffaella
R
Dipartimento di Scienze Biomolecolari e Biotecnologie, Università di Milano, Via Celoria 26, 20133 Milano, Italy.
Mantovani
Roberto
R
D023081:CCAAT-Binding Factor / Q000378:metabolism*; D002453:Cell Cycle / Q000235:genetics; D002460:Cell Line; D047369:Chromatin Immunoprecipitation / Q000379:methods; D056970:Co-Repressor Proteins; D019926:Cyclin B / Q000235:genetics; D056765:Cyclin B2; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018816:Genes, cdc; D056466:Histone Demethylases; D006657:Histones / Q000378:metabolism*; D006801:Humans; D008745:Methylation; D008836:Micrococcal Nuclease; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009707:Nucleosomes / Q000737:chemistry / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics; D011401:Promoter Regions, Genetic; D011499:Protein Processing, Post-Translational; D012097:Repressor Proteins / Q000378:metabolism*
D023081:CCAAT-Binding Factor; C503758:CCNB2 protein, human; D056970:Co-Repressor Proteins; D019926:Cyclin B; D056765:Cyclin B2; D004268:DNA-Binding Proteins; D006657:Histones; D009419:Nerve Tissue Proteins; D009707:Nucleosomes; D018809:Proliferating Cell Nuclear Antigen; C121019:RCOR1 protein, human; D012097:Repressor Proteins; D056466:Histone Demethylases; C495195:KDM1A protein, human; D010089:Oxidoreductases, N-Demethylating; D008836:Micrococcal Nuclease